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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-03-20 to 2014-10-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis.
The concentration of boron in the test preparations was verified by chemical analysis at 0 and 72 hours.
Total Organic Carbon (TOC) analysis was performed on the test solutions containing no algal cells at 0 and 72 hours.
Samples were frozen prior to analysis.
Duplicate samples were taken and frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
Preliminary media preparation trial
Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation.
The test item was categorised as being a "difficult substance" as defined by the OECD.

Range-finding test

Nominal amounts of test item (20 and 200 mg) were each separately added to the surface of 2 litres of culture medium to give 10 and 100 mg/L loading rates respectively. After the addition of the test item the water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour prior to the removal of any undissolved test item by filtration through a glass wool plug. A wide bore glass tube covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the aqueous phase removed by mid-depth siphoning (the first 75 - 100 mL discarded) to give the 10 and 100 mg/L loading rates. Microscopic observations of the test preparations were performed after filtering and showed there to be no undissolved test item present.
An aliquot (500 mL) of each of the stock solutions was separately inoculated with algal suspension (4.9 mL) to give the required test concentrations of 10 and 100 mg/L.

A second range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal loading rates of 0.010, 0.10 and 1.0 mg/L for a period of 72 hours.
The test samples were prepared as above using 20 mg of the test material in 20 L culture medium to give the 1.0 mg/L loading rate. A series of dilutions were made from the 1.0 mg/L stock solution to give further stock solutions of 0.10 and 0.010 mg/L.
An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (5.2 mL) to give the required test concentrations of 0.010, 0.10 and 1.0 mg/L.

Definitive test
The test samples were prepared as above using 250 mg of the test material in 25L culture medium to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further stock solutions of 32, 10, 3.2 and 1.0% v/v saturated solution.
An aliquot (500 mL) of each of the stock solutions was separately inoculated with 2.9 mL of algal suspension to give the required test concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4.
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland
Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1 °C.
ACCLIMATION
Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10E03 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10E04 - 10E05 cells/mL.

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None
Hardness:
Not specified
Test temperature:
24 ± 1 ºC
pH:
7.6 - 8.1
Dissolved oxygen:
Not measured
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations
1st Range finder: 10 and 100 mg/L
2nd Range finder: 0.010, 0.10 and 1.0 mg/L
Definitive test: 1.0, 3.2, 10, 32 and 100% v/v saturated solution
Details on test conditions:
Range finding tests
The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were used for each control and test concentration.
The control group was maintained under identical conditions but not exposed to the test item.
At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
The results of the initial range-finding test showed significant inhibition of growth occurred at all test loading rates and so a second range-finding test was conducted. Exposure conditions in the second range-finding test were the same as those in the initial test.
Definitive test
As in the range-finding tests 250 mL glass conical flasks were used. Six flasks each containing 100 mL of solution were used for the control and three flasks each containing 100 mL were used for each treatment group.
The control group was maintained under identical conditions but not exposed to the test item.
Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 8.68E05 cells per mL. Inoculation of 500 mL of test medium with 2.9 mL of this algal suspension gave an initial nominal cell density of 5E03 cells per mL and had no significant dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
Reference substance (positive control):
yes
Remarks:
potassium dichromate at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
9 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
carbon content
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
5 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
carbon content
Basis for effect:
other: yield
Details on results:
Range-finding Tests
The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the range-finding tests can be found in the attached document.
The results showed no effect on growth rate at the loading rates of 0.010, 0.10 and 1.0 mg/L. However, growth was observed to be reduced at 10 and 100 mg/L.
Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution were selected for the definitive test.
Chemical analysis of the 1.0 mg/L loading rate test preparations at 0 and 72 hours showed measured boron concentrations of 0.024 and 0.057 mg/L respectively were obtained. Analysis of the 100 mg/L loading rate test preparation at 0 hours showed a measured boron concentration of 2.1 mg/L was obtained. Due to a technical oversight a sample of the 100 mg/L loading rate was not taken for chemical analysis at 72 hours.

Definitive Test
Verification of Test Concentrations
Chemical analysis of the test preparations at 0 and 72 hours showed measured boron concentrations to range from 0.070 to 2.5 mg/L. A measured boron concentration of 0.27 mg/L was observed in both the original and duplicate control samples at 72 hours. Given that a measured concentration of less than the LOQ was obtained at the start of the test it was considered that this result was due to contamination from an unknown source during the sampling process at 72 hours. As such, this result was considered to have had no adverse effect on the outcome of the test.
Total Organic Carbon Analysis
Total Organic Carbon (TOC) analysis of test preparations at 0 and 72 hours showed measured carbon concentrations to range from less than the limit of quantification (LOQ), determined to be 1.0 mg C/L at 1.0% v/v saturated solution, through to 32 mg/L at 100% v/v saturated solution (equivalent to test item concentrations of less than the LOQ to 42 mg/L based on a test item carbon content of 75.78%).
It was considered appropriate to calculate the results based on the equivalent test item concentrations obtained from TOC analysis as these results give an estimate of the total dissolved test item concentrations present.
Growth Data
The growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test item over the 72-Hour exposure period.
Accordingly the following results were determined from the data:
Inhibition of Growth Rate
ErC10 (0 - 72 h) : 4.4 mg/L
ErC20 (0 - 72 h) : 5.8 mg/L
ErC50 (0 - 72 h) : 9.0 mg/L; 95% confidence limits 7.8 - 11 mg/L
where ErCx is the test concentration that reduced growth rate by x%.
Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955). There were no statistically significant differences between the control, 1.0 and 3.2% v/v saturated solution test concentrations (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 3.2% v/v saturated solution, equivalent to a test item concentration of 1.9 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 10% v/v saturated solution, equivalent to a test item concentration of 5.1 mg/L.
Inhibition of Yield
EyC10 (0 - 72 h) : 2.7 mg/L
EyC20 (0 - 72 h) : 3.4 mg/L
EyC50 (0 - 72 h) : 5.0 mg/L; 95% confidence limits 4.4 – 5.7 mg/L
There were no statistically significant differences between the control, 1.0 and 3.2% v/v saturated solution test concentrations (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 3.2% v/v saturated solution, equivalent to a test item concentration of 1.9 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 10% v/v saturated solution, equivalent to a test item concentration of 5.1 mg/L.
Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 124 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.
Mean cell density of control at 0 hours : 5.67E03 cells per mL
Mean cell density of control at 72 hours : 7.06E05 cells per mL
The mean coefficient of variation for section by section specific growth rate for the control cultures was 10% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.
The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 1% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.
Observations on Cultures
All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 1.0, 3.2 and 10% v/v saturated solution, however cell debris was observed to be present in the test cultures at 32 and 100% v/v saturated solution.
Water Quality Criteria
The pH value of the control cultures was observed to increase from pH 7.5 at 0 hours to pH 8.0 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.
Observations on Test Item Solubility
Observations on the test media were carried out during the mixing and testing of the saturated solution.
At the both the start and end of the mixing period, and following a 1-Hour standing period, the 100% v/v saturated solution was observed to have formed a clear colourless media column with test item on the surface. Microscopic examination after filtering showed the glass wool plug had removed all of the undissolved test item present.
At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control, 1.0, 3.2 and 10% v/v saturated solution test cultures were observed to be green dispersions whilst the 32 and 100% v/v saturated solution test cultures were observed to be clear colourless solutions.
Results with reference substance (positive control):
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 – 72 h) : 1.2 mg/L; 95% confidence limits 1.1 – 1.4 mg/L
EyC50 (0 – 72 h) : 0.63 mg/L; 95% confidence limits 0.57 – 0.70 mg/L
No Observed Effect Concentration (NOEC) based on growth rate: 0.50 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 1.0 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L
The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

See attached document

Validity criteria fulfilled:
yes
Conclusions:
Exposure of the test item to Pseudokirchneriella subcapitata gave a 72-Hour EC50 value based on growth rate of 9.0 mg/L (95% CI = 7.8 -11 mg/L). The No Effect Concentration was 1.9 mg/L and the LOEC was considered to be 5.1 mg/L.
Executive summary:

Introduction

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

Methods

Due to the low aqueous solubility and pure nature of the test item, for the purposes of the definitive test, the test medium was prepared as a slow stir saturated solution. Following preliminary range-finding tests, Pseudokirchneriella subcapitata was exposed to solutions of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. The test item solutions were prepared by stirring an excess (100 mg/L) of test item in culture medium using a magnetic stirrer at a rate such that a dimple was formed at the water surface for 23 hours. After the stirring period, the saturated solution was allowed to stand for 1-Hour prior to the removal of any undissolved test item by filtration through a glass wool plug (first approximate 75-100 mL discarded) to produce a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test groups. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Results

Chemical analysis of the test preparations at 0 and 72 hours showed measured boron concentrations to range from 0.070 to 2.5 mg/L. Total Organic Carbon (TOC) analysis of test preparations at 0 and 72 hours showed measured carbon concentrations to range from less than the limit of quantification (LOQ), determined to be 1.0 mg C/L at 1.0% v/v saturated solution, through to 32 mg/L at 100% v/v saturated solution (equivalent to test item concentrations of less than the LOQ to 42 mg/L based on a test item carbon content of 75.78%). It was considered appropriate to calculate the results based on the equivalent test item concentrations obtained from TOC analysis as these results give an estimate of the total dissolved test item concentrations present. Exposure of the test item to Pseudokirchneriella subcapitata gave a 72-Hour EC50 value based on growth rate of 9.0 mg/L (95% CI = 7.8 -11 mg/L). The No Effect Concentration was 1.9 mg/L and the LOEC was considered to be 5.1 mg/L.

Description of key information

Exposure of the test item to Pseudokirchneriella subcapitata gave a 72-Hour EC50 value based on growth rate of 9.0 mg/L (95% CI = 7.8 -11 mg/L). The No Effect Concentration was 1.9 mg/L and the LOEC was considered to be 5.1 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
9 mg/L
EC10 or NOEC for freshwater algae:
1.9 mg/L

Additional information

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

Due to the low aqueous solubility and pure nature of the test item, for the purposes of the definitive test, the test medium was prepared as a slow stir saturated solution. Following preliminary range-finding tests, Pseudokirchneriella subcapitata was exposed to solutions of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. The test item solutions were prepared by stirring an excess (100 mg/L) of test item in culture medium using a magnetic stirrer at a rate such that a dimple was formed at the water surface for 23 hours. After the stirring period, the saturated solution was allowed to stand for 1-Hour prior to the removal of any undissolved test item by filtration through a glass wool plug (first approximate 75-100 mL discarded) to produce a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test groups. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Chemical analysis of the test preparations at 0 and 72 hours showed measured boron concentrations to range from 0.070 to 2.5 mg/L. Total Organic Carbon (TOC) analysis of test preparations at 0 and 72 hours showed measured carbon concentrations to range from less than the limit of quantification (LOQ), determined to be 1.0 mg C/L at 1.0% v/v saturated solution, through to 32 mg/L at 100% v/v saturated solution (equivalent to test item concentrations of less than the LOQ to 42 mg/L based on a test item carbon content of 75.78%). It was considered appropriate to calculate the results based on the equivalent test item concentrations obtained from TOC analysis as these results give an estimate of the total dissolved test item concentrations present. Exposure of the test item to Pseudokirchneriella subcapitata gave a 72-Hour EC50 value based on growth rate of 9.0 mg/L (95% CI = 7.8 -11 mg/L). The No Effect Concentration was 1.9 mg/L and the LOEC was considered to be 5.1 mg/L.